A composite polypeptide external dressing and its application in promoting skin wound healing

Abstract

The application provides a kind of composite polypeptide external application dressings and its application in promoting skin wound healing, by polypeptide synergistic effect and intelligent carrier design realizes efficient healing promotion.The dressing is composed of proliferation-promoting peptides, antibacterial and anti-inflammatory peptides, collagen synthesis promoting peptides and penetration-promoting peptides in a specific ratio, supplemented with natural antioxidants and metal chelating agents, and carboxymethyl chitosan-chondroitin sulfate temperature-sensitive hydrogel is used as the carrier.Preparation method realizes polypeptide activity protection and function strengthening through directional enzymolysis, molecular weight cut-off, chemical conjugation and other technologies, combined with ultrasonic dispersion and crosslinking process to improve the stability of the dressing.The dressing can synergistically regulate the whole cycle of wound healing, improve the penetration and stability of active ingredients.The effect verification shows that the wound healing rate is 96.7% in 14 days, the concentration of inflammatory factors is low, the collagen content is high, and it is suitable for preparing skin wound healing drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a composite polypeptide topical dressing and its application in promoting skin wound healing. Background Technology

[0002] Skin wound healing is a complex physiological repair process involving multiple stages such as inflammatory response, cell proliferation, matrix synthesis and remodeling, and is widely observed in clinical settings such as trauma, burns, chronic ulcers, and post-surgical procedures. Delayed or poor wound healing not only increases patient suffering and prolongs treatment, but can also lead to complications such as infection, scar hyperplasia, and even tissue necrosis, posing significant challenges to medical care. Therefore, the development of topical dressings that combine antibacterial and anti-inflammatory properties, promote cell proliferation, accelerate collagen synthesis, and enhance the penetration efficiency of active ingredients has become a research hotspot in the biomedical field.

[0003] Currently, commonly used wound dressings in clinical practice are mainly divided into two categories: traditional dressings and novel biological dressings. Traditional dressings, such as gauze and cotton pads, while possessing basic functions of absorbing exudate and providing physical protection, have significant drawbacks such as poor moisture retention, easy adhesion to the wound surface, potential for secondary damage during dressing changes, and inability to inhibit infection. They are no longer sufficient to meet the repair needs of complex wounds.

[0004] To overcome the shortcomings of traditional dressings, novel biological dressings are constantly emerging, among which peptide dressings have attracted much attention due to their advantages such as good biocompatibility, high activity, and few side effects. However, existing peptide dressings still have many problems that need to be solved: First, their functions are limited, with most products focusing only on a single repair stage. For example, single antimicrobial peptides can only inhibit microbial growth, and single growth factor peptides can only promote cell proliferation, lacking the ability to synergistically regulate the entire wound healing cycle, resulting in limited overall repair effects. Second, the active ingredients have poor permeability. The barrier effect of the stratum corneum makes it difficult for peptide molecules to effectively penetrate and reach the deep layers of the wound to exert their effects, significantly reducing the actual efficacy of the dressing. Third, they lack stability. Peptide components are easily deactivated by factors such as temperature and enzymatic hydrolysis, leading to a decrease in activity during dressing storage and affecting clinical application effects. Fourth, the carrier matrix of some dressings has poor biocompatibility and has not formed an effective loading and sustained-release system for active peptides, further weakening the repair effect.

[0005] In the application of peptide raw materials, existing technologies also have significant limitations. For example, proliferation-promoting components mostly rely on chemical synthesis or single plant extracts, making it difficult to balance activity and safety; antimicrobial peptides have limited sources and narrow antimicrobial spectrums, making it difficult to cope with the complex microbial environment of wounds; collagen synthesis promoters are mostly intact collagen proteins with large molecular weights, making them difficult to absorb, and their degradation rate does not match the pace of wound repair; penetration-enhancing components often use chemical reagents, resulting in poor biocompatibility and potential irritation to the wound. Furthermore, existing technologies generally neglect the synergistic effects between different peptide components, as well as the impact of excipient selection and preparation processes on the overall performance of dressings, making it difficult for products to achieve ideal wound healing effects in clinical applications.

[0006] In summary, to address the current problems of single-function topical dressings, poor penetration of active ingredients, insufficient stability, and lack of synergistic effects, we have developed a composite peptide topical dressing that combines multiple functional peptides with an excellent carrier matrix, possessing highly efficient antibacterial and anti-inflammatory properties, promotes proliferation and collagen synthesis, and has strong penetration capabilities. This has significant clinical value and application prospects. Summary of the Invention

[0007] In view of this, the present invention proposes a composite polypeptide topical dressing and its application in promoting skin wound healing, thereby solving the above problems.

[0008] The technical solution of the present invention is implemented as follows: a compound polypeptide external dressing, comprising the following raw materials in parts by weight: 20-40 parts of proliferating peptide, 15-35 parts of antibacterial and anti-inflammatory peptide, 10-30 parts of collagen synthesis promoting peptide, and 5-8 parts of penetration promoting peptide.

[0009] Furthermore, it includes the following raw materials in parts by weight: 30 parts of growth-promoting peptide, 25 parts of antibacterial and anti-inflammatory peptide, 20 parts of collagen synthesis-promoting peptide, and 7 parts of penetration-promoting peptide.

[0010] Furthermore, the proliferation-promoting peptide is a Dendrobium officinale stem tip regeneration peptide, and its extraction method specifically includes:

[0011] (1) Raw material pretreatment: Take fresh Dendrobium officinale stem tip meristem (length ≤5mm, surface disinfected with 75% ethanol for 30 seconds, disinfected with 0.1% mercuric chloride solution for 5 minutes, and rinsed with sterile water 3 times), place it in a dark environment at 4℃ for 24-72 hours to activate the expression of meristem regeneration-related genes and obtain pretreated raw material;

[0012] (2) Combined enzymatic hydrolysis: After the pretreated raw materials are chopped, they are dispersed in deionized water at a material-to-liquid ratio of 1:(10-15 g / mL). A mixed enzyme of cellulase (activity ≥100,000 U / g) and pectinase (activity ≥50,000 U / g) is added at a mass ratio of 1:1-2. The total amount added is 0.5%-1.0% of the dry weight of the raw materials. The pH of the system is adjusted to 4.5-5.5. The enzyme is hydrolyzed at a constant temperature of 40-50℃ for 6-8 hours (stirred once every 2 hours during the enzymatic hydrolysis at a speed of 100-150 rpm).

[0013] (3) Enzyme inactivation and crude separation: After the enzymatic hydrolysis is completed, the temperature is raised to 95℃ and held for 10 minutes to inactivate the enzyme. After cooling, the supernatant is centrifuged (4000 rpm, 10 minutes) and the supernatant is collected. The unhydrolyzed macromolecular polysaccharides and proteins are removed by ultrafiltration (molecular weight cutoff 1.2 kDa). The filtrate with a molecular weight of 800-1200 Da is collected, purified and dried to obtain white powdered Dendrobium officinale stem tip regeneration peptide with a purity ≥90% and a molecular weight distribution of 800-1200 Da.

[0014] Furthermore, the antibacterial and anti-inflammatory peptide is derived from short disulfide-containing peptides obtained by enzymatic hydrolysis of byssal mucin from marine mollusks, with a molecular weight ≤1.2kDa. The extraction method specifically includes:

[0015] (1) Raw material pretreatment: Take fresh mussels (Mytilusedulis) byssal threads (filamentous structures attached to the inside of the shell), rinse to remove surface mud and microorganisms, cut into small pieces and disperse in Tris-HCl buffer at a material-to-liquid ratio of 1:(8-10) g / mL, add protease inhibitor (1mM benzyl sulfonyl fluoride), sonicate at 4℃ (power 200W, frequency 40kHz, time 10 minutes), centrifuge (8000rpm, 15 minutes), collect the precipitate, which is crude byssal adhesive protein;

[0016] (2) Targeted enzymatic hydrolysis: The crude byssal mucin was redispersed in deionized water at a substrate concentration of 3%-5% w / v. Papain (activity ≥80,000 U / g, preferably from papaya fruit) was added at a concentration of 0.4%-0.6% of the substrate mass. The pH of the system was adjusted to 6.0-6.5, and the enzyme was hydrolyzed at 42-45℃ for 3-4 hours. After the enzymatic hydrolysis was completed, the temperature was raised to 80-100℃ and held for 8-12 minutes to inactivate the enzyme. After cooling, the supernatant was collected by centrifugation (5000 rpm, 10 minutes), ultrafiltration, purification, and freeze-drying to obtain a white powder.

[0017] Furthermore, the collagen synthesis promoting peptide is derived from bovine bone type I collagen. The extraction method is as follows: Bovine bone type I collagen is crushed, 5-8 times the amount of deionized water is added, the pH is adjusted to 7.0-7.5, 0.6-1.0% of collagen protein mass of protease is added for enzymatic hydrolysis for 2-4 hours, after enzyme inactivation, it is purified by 1000-2000 Da ultrafiltration membrane, and dried to obtain glycine-proline enriched tripeptide.

[0018] Furthermore, the permeation-enhancing peptide is a menthol-peptide conjugate extracted from peppermint leaves. Its preparation method specifically includes: pulverizing peppermint leaves and passing them through a 60-80 mesh sieve, adding 10-15 times the weight of deionized water, adding 0.3%-0.8% of a compound enzyme (based on the weight of peppermint leaves), and enzymatically hydrolyzing at 40-45℃ for 1.5-2.5 hours. The compound enzyme is a 1:1-3 mass ratio of cellulase and protease. Centrifuging is performed to obtain the supernatant as a peppermint peptide solution. The peppermint peptide solution is mixed with menthol at a mass ratio of 5-8:1, and 0.1%-0.3%wt of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is added to the mixture. The mixture is reacted at 30-35℃ for 1-3 hours, adsorbed through macroporous resin, filtered through a 300-800 Da ultrafiltration membrane, and then freeze-dried to obtain the menthol-peptide conjugate.

[0019] Furthermore, it also includes 0.2-0.6 parts of a natural antioxidant and 0.1-0.3 parts of a metal ion chelating agent; the natural antioxidant is selected from tea polyphenols, rosmarinic acid or total asiaticoside, and the metal ion chelating agent is EDTA-2Na or sodium citrate.

[0020] This invention also provides a method for preparing a composite polypeptide topical dressing, comprising the following steps:

[0021] S1. Raw material pretreatment and dissolution: Weigh the proliferation-promoting peptide, antibacterial and anti-inflammatory peptide, collagen synthesis-promoting peptide, and permeation-promoting peptide, add sterile deionized water, and stir at low speed for 10-15 minutes at 25-30℃ until completely dissolved to obtain a single-component solution of each peptide.

[0022] S2. Preparation of carrier matrix: Mix carboxymethyl chitosan and chondroitin sulfate at a mass ratio of (2-4):1, dissolve in deionized water at 40-50℃, and stir at 200-300 rpm until completely dissolved to form carrier mother liquor;

[0023] S3. Assembly of the composite system: The single-component peptide solution obtained in S1 and the antioxidant and chelating agent are added sequentially to the carrier mother liquor in S2, ultrasonically dispersed for 15-25 minutes, pH adjusted to 6.8-7.2, and allowed to stand for cross-linking for 3-5 hours;

[0024] S4. Sterilization and Packaging: The cross-linked system is sterilized by γ-ray irradiation and then packaged to obtain the composite polypeptide external dressing.

[0025] Furthermore, in step S1, the ratio of peptide to water in the liquid to the liquid is 1:10-15 g:mL, and the stirring speed is 100-150 rpm.

[0026] In step S3, the ultrasonic power is 400-600W, the static crosslinking temperature is 25-30℃, the solid content of the crosslinked system is 15-25wt%, and the pH value is 5.5-6.5.

[0027] The gamma ray irradiation dose in step S4 is 15-25 kGy, and the irradiation time is 10-20 minutes.

[0028] The application of the compound polypeptide topical dressing in the preparation of drugs that promote skin wound healing.

[0029] Compared with the prior art, the beneficial effects of the present invention are:

[0030] This invention significantly improves the efficiency and safety of skin wound repair through the synergistic effect of compound peptides. Among them, proliferative peptides stimulate fibroblast proliferation, antibacterial and anti-inflammatory peptides inhibit the release of inflammatory factors, collagen synthesis promotes peptides to accelerate matrix reconstruction, and permeation-promoting peptides enhance the transdermal efficiency of active ingredients, forming a closed loop of anti-inflammatory, proliferative, and reconstruction effects. Active peptides are extracted using bio-enzymatic hydrolysis and molecular weight cutoff technology, with a purity ≥90%. Combined with a carboxymethyl chitosan-chondroitin sulfate thermosensitive gel carrier, pH-responsive adhesion and sustained drug release are achieved. After the dressing is stored at 4°C for 30 days, the activity retention rate is still higher than 91%, avoiding the defects of activity loss and poor permeability. The dual protection system of natural antioxidants and EDTA-2Na inhibits the oxidative degradation of peptides. The ultrasonic dispersion and static cross-linking technology in the process makes the dressing structure dense and uniform, effectively avoiding the burst release effect. Detailed Implementation

[0031] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0032] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0034] Example 1

[0035] The compound polypeptide topical dressing comprises the following raw materials in parts by weight: 20 parts of proliferation-promoting peptide, 15 parts of antibacterial and anti-inflammatory peptide, 10 parts of collagen synthesis-promoting peptide, 5 parts of penetration-promoting peptide, 0.2 parts of natural antioxidant, and 0.1 parts of metal ion chelating agent.

[0036] The proliferation-promoting peptide is a regeneration peptide from the stem tips of Dendrobium officinale, and its extraction method specifically includes:

[0037] (1) Raw material pretreatment: Take fresh Dendrobium officinale stem tip meristem (length ≤5mm, surface disinfected with 75% ethanol for 30 seconds, disinfected with 0.1% mercuric chloride solution for 5 minutes, and rinsed with sterile water 3 times), place it in a dark environment at 4℃ for 48 hours to induce culture, activate the expression of meristem regeneration-related genes, and obtain pretreated raw material;

[0038] (2) Combined enzymatic hydrolysis: After the pretreated raw materials are chopped, they are dispersed in deionized water at a material-to-liquid ratio of 1:12 (g / mL). A mixture of cellulase (activity ≥100,000 U / g) and pectinase (activity ≥50,000 U / g) is added at a mass ratio of 1:2. The total amount added is 0.8% of the dry weight of the raw materials. The pH of the system is adjusted to 5, and the enzymatic hydrolysis is carried out at 45℃ for 7 hours (stirred once every 2 hours during the enzymatic hydrolysis at a speed of 130 rpm).

[0039] (3) Enzyme inactivation and crude separation: After the enzymatic hydrolysis, the temperature is raised to 95℃ and held for 10 minutes to inactivate the enzyme. After cooling, the supernatant is centrifuged (4000 rpm, 10 minutes) and the supernatant is collected. The supernatant is then ultrafiltered (with a molecular weight cutoff of 1.2 kDa) to remove unhydrolyzed macromolecular polysaccharides and proteins. The filtrate with a molecular weight of 1000 Da is collected, purified, and dried to obtain white powdered Dendrobium officinale stem tip regeneration peptide with a purity of ≥90% and a molecular weight distribution of 1000 Da.

[0040] The antibacterial and anti-inflammatory peptides are derived from short disulfide-containing peptides obtained by enzymatic hydrolysis of byssal mucin from marine mollusks, with a molecular weight ≤1.2kDa. The extraction method specifically includes:

[0041] (1) Raw material pretreatment: Take fresh mussel byssal threads (filamentous structures attached to the inside of the shell), rinse to remove surface mud and microorganisms, cut into small pieces and disperse in pH 7.0 Tris-HCl buffer at a material-to-liquid ratio of 1:9 g / mL, add protease inhibitor (1mM benzyl sulfonyl fluoride), sonicate at 4℃ (power 200W, frequency 40kHz, time 10 minutes), centrifuge (8000rpm, 15 minutes), collect the precipitate, which is crude byssal adhesive protein;

[0042] (2) Targeted enzymatic hydrolysis: The crude byssal mucin was redispersed in deionized water at a substrate concentration of 4% w / v. Papain (activity ≥ 80,000 U / g, selected from papaya fruit) was added at a concentration of 0.5% of the substrate mass. The pH of the system was adjusted to 6.0, and the enzyme was hydrolyzed at 45°C for 3 hours. After the enzymatic hydrolysis was completed, the temperature was raised to 100°C and held for 10 minutes to inactivate the enzyme. After cooling, the supernatant was collected by centrifugation (5000 rpm, 10 minutes), ultrafiltration, purification, and freeze-drying to obtain a white powder.

[0043] The collagen synthesis promoting peptide is derived from bovine bone type I collagen. The extraction method is as follows: Bovine bone type I collagen is crushed, 7 times the amount of deionized water is added, the pH is adjusted to 7.2, 0.8% of collagen protein mass of protease is added for enzymatic hydrolysis for 3 hours, after enzyme inactivation, it is purified by 1500 Da ultrafiltration membrane and dried to obtain glycine-proline enriched tripeptide.

[0044] The permeation-enhancing peptide is a menthol-peptide conjugate extracted from peppermint leaves. Its preparation method specifically includes: pulverizing peppermint leaves and passing them through a 70-mesh sieve, adding 12 times the weight of deionized water, adding 0.5% of a compound enzyme (based on the weight of peppermint leaves), and enzymatically hydrolyzing at 42°C for 2 hours. The compound enzyme is a 1:2 mass ratio of cellulase and protease. The supernatant is collected by centrifugation to obtain a peppermint peptide solution. The peppermint peptide solution is mixed with menthol at a mass ratio of 7:1, and 0.2% wt of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is added to the mixture. The mixture is reacted at 32°C for 2 hours, adsorbed through macroporous resin, filtered through a 500 Da ultrafiltration membrane, and then freeze-dried to obtain the menthol-peptide conjugate.

[0045] The natural antioxidant is selected from tea polyphenols, and the metal ion chelating agent is EDTA-2Na.

[0046] Example 2

[0047] The compound polypeptide topical dressing comprises the following raw materials in parts by weight: 40 parts of proliferation-promoting peptide, 35 parts of antibacterial and anti-inflammatory peptide, 30 parts of collagen synthesis-promoting peptide, 8 parts of penetration-promoting peptide, 0.6 parts of natural antioxidant, and 0.3 parts of metal ion chelating agent;

[0048] The proliferation-promoting peptide, antibacterial and anti-inflammatory peptide, collagen synthesis-promoting peptide, and permeation-promoting peptide were prepared using the method described in Example 1.

[0049] The natural antioxidant is selected from rosmarinic acid, and the metal ion chelating agent is EDTA-2Na.

[0050] Example 3

[0051] The compound polypeptide topical dressing comprises the following raw materials in parts by weight: 30 parts of proliferation-promoting peptide, 25 parts of antibacterial and anti-inflammatory peptide, 20 parts of collagen synthesis-promoting peptide, and 7 parts of penetration-promoting peptide.

[0052] The proliferation-promoting peptide, antibacterial and anti-inflammatory peptide, collagen synthesis-promoting peptide, and permeation-promoting peptide were prepared using the method described in Example 1.

[0053] The natural antioxidant is selected from total glycosides of Centella asiatica, and the metal ion chelating agent is sodium citrate.

[0054] The dressings in Examples 1-3 above were prepared using the following method:

[0055] S1. Raw material pretreatment and dissolution: Weigh the proliferation-promoting peptide, antibacterial and anti-inflammatory peptide, collagen synthesis-promoting peptide, and permeation-promoting peptide, add sterile deionized water, and the ratio of peptide to water in g:mL = 1:12. Stir at 30℃ at low speed of 120rpm for 12 minutes until completely dissolved to obtain a single-component solution of each peptide.

[0056] S2. Preparation of carrier matrix: Carboxymethyl chitosan and chondroitin sulfate are mixed at a mass ratio of 3:1, dissolved in deionized water at 45℃, and stirred at 250rpm until completely dissolved to form carrier mother liquor.

[0057] S3. Assembly of the composite system: The single-component solution of the polypeptide obtained in S1 and the antioxidant and chelating agent were added to the carrier mother liquor in S2 in sequence, ultrasonically dispersed at 500W for 20 minutes, pH was adjusted to 7, and crosslinked for 4 hours. The crosslinking temperature was 28℃. The solid content of the system after crosslinking was 20wt%, and the pH value was 6.

[0058] S4. Sterilization and Packaging: The cross-linked system is sterilized by γ-ray irradiation with a dose of 20 kGy for 15 minutes, and then packaged to obtain the composite polypeptide external dressing.

[0059] Comparative Example 1

[0060] The difference between this comparative example and Example 3 is that the composite polypeptide topical dressing lacks proliferative peptides.

[0061] Comparative Example 2

[0062] The difference between this comparative example and Example 3 is that the composite polypeptide topical dressing lacks antibacterial and anti-inflammatory peptides.

[0063] Comparative Example 3

[0064] The difference between this comparative example and Example 3 is that the composite polypeptide topical dressing lacks a penetration-enhancing peptide.

[0065] Comparative Example 4

[0066] The difference between this comparative example and Example 3 is that the carrier is replaced with an equal mass of ordinary chitosan in the preparation process, while the rest is the same as in Example 3.

[0067] Comparative Example 5

[0068] The difference between this comparative example and Example 3 is that the preparation process does not involve ultrasonic dispersion and static cross-linking; the mixture is directly mixed and then sterilized.

[0069] Effect verification experiment

[0070] I. Experimental Materials and Subjects

[0071] 1. Test samples: Composite polypeptide topical dressings (gel formulation) prepared in Examples 1, 2 and 3; control dressings prepared in Comparative Examples 1-5; blank control was sterile physiological saline.

[0072] 2. Experimental animals: SPF-grade female SD rats, weighing 220-250g, a total of 80 rats (10 rats per group, divided into 8 groups: Example 1-3 groups, Comparative 1-5 groups, and blank control group).

[0073] II. Test Methods

[0074] 1. Wound model establishment

[0075] After 3 days of acclimatization, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (3 mL / kg), their backs were shaved and disinfected with iodine, and their backs were scalded with an 80℃ constant temperature water bath scalding device for 15 seconds to create deep second-degree burn wounds (pathological section verification: full-thickness epidermal necrosis, superficial dermal damage, and residual hair follicles and sweat glands). The wound area was uniformly 2cm×2cm.

[0076] 2. Grouping and Dosing

[0077] Postoperatively, the animals were randomly divided into 8 groups of 10 each. Dressings were changed once daily, and the procedure was as follows:

[0078] Examples 1-3: Apply the corresponding dressing (0.5g / wound) to the wound and cover with sterile gauze for fixation.

[0079] Comparative Examples 1-5: Apply the corresponding control dressing (0.5g / wound), and fix it in the same way as above.

[0080] Blank control group: Sterile saline (0.5 mL / wound) was applied to the wound and fixed in the same way as above.

[0081] The dressing change cycle was 14 days, during which the rats' mental state, wound exudation, and infection status were observed.

[0082] 3. Indicator detection time points

[0083] Samples were taken and tested on the 3rd, 7th and 14th day after surgery, and stability was tested earlier after sample preparation.

[0084] 4. Detection Indicators and Methods

[0085] (1) Wound healing rate

[0086] Methods: Before each dressing change, the wound outline was traced with a sterile transparent membrane, photographed, and the wound area was calculated using ImageJ software. The healing rate was then calculated using the formula:

[0087] Wound healing rate (%) = (Initial wound area - Unhealed wound area) / Initial wound area × 100%

[0088] Testing time: 3 days, 7 days, and 14 days after surgery.

[0089] (2) Concentration of inflammatory factors in the wound (IL-6, TNF-α)

[0090] Methods: Seven days after surgery, three rats were taken from each group, and wound exudate was aseptically collected. The supernatant was collected by centrifugation (3000 rpm, 10 minutes), and the concentrations of IL-6 and TNF-α were detected by ELISA kit according to the instructions.

[0091] (3) Collagen content in the wound (hydroxyproline method)

[0092] Methods: 14 days after surgery, 3 rats were taken from each group, and 0.5g of the central tissue of the wound was taken. The hydroxyproline content in the tissue was detected by the hydroxyproline detection kit (hydroxyproline accounts for 13.4% of collagen, which is converted into collagen content).

[0093] (4) Antibacterial effect on wound (colony count)

[0094] Methods: Three days post-surgery, three rats were taken from each group. Exudate from the wound was collected using a sterile cotton swab and spread onto LB agar medium. After incubation at 37°C for 24 hours, the colony count (CFU / cm³) was determined using a colony counter. 2 (5) Dressing stability (retention rate of active peptides)

[0095] Methods: After sample preparation, the samples were sealed and stored at 4℃ for 30 days. High-performance liquid chromatography (HPLC) was used to detect the content of proliferative peptides and antibacterial and anti-inflammatory peptides in the dressing, and the activity retention rate was calculated.

[0096] Activity retention rate (%) = (Peptide content after storage / Initial peptide content) × 100%

[0097] III. Test Results

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] Example 3 showed the best performance in all indicators: the 14-day healing rate reached 96.7%, which was significantly higher than other groups; the concentration of inflammatory factors was the lowest, the collagen content was the highest, the number of colonies was the fewest, and the stability was the best (activity retention rate ≥94%), proving that this ratio is the optimal combination of tetrapeptide synergy, excipient compatibility, and process optimization.

[0104] The healing rate of Comparative Example 1 was significantly lower than that of Example 3, with a decrease in collagen content of 42.3%, indicating that Dendrobium officinale stem tip regeneration peptide can effectively promote fibroblast proliferation and collagen synthesis, which is the core of accelerating wound repair.

[0105] Compared with Example 3, Comparative Example 2 showed an increase in bacterial colony count, a 1.5-2.3-fold increase in inflammatory factor concentration, and a 22.3% decrease in healing rate, demonstrating that the antibacterial and anti-inflammatory effects of mussel foot mucin protease hydrolysate peptides can reduce wound infection, alleviate inflammatory response, and create a favorable environment for healing.

[0106] The healing rate of Comparative Example 3 was lower than that of Example 3 because the lack of menthol-peptide conjugate led to a decrease in peptide penetration efficiency, and the active ingredients could not effectively reach the deep layers of the wound, thus weakening the repair effect.

[0107] The healing rate of Comparative Example 4 decreased by 11.9%, and the activity retention rate decreased by 14-15%. This is because the carboxymethyl chitosan-chondroitin sulfate composite carrier has better biocompatibility, moisturizing properties, and peptide loading capacity than ordinary chitosan, which can prolong peptide activity.

[0108] The healing rate of Comparative Example 5 decreased by 14.8%, and the activity retention rate decreased by 18-19%. This is because ultrasonic dispersion can promote uniform mixing of peptides and carriers, and static cross-linking enhances the structural stability of the dressing and avoids loss of active ingredients.

[0109] The blank control group had the worst results in all indicators, further proving that the dressing of the present invention can significantly promote wound healing, and that the synergistic effect of each component and process is indispensable.

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composite polypeptide external dressing, characterized in that: It includes the following raw materials in parts by weight: 20-40 parts of growth-promoting peptides, 15-35 parts of antibacterial and anti-inflammatory peptides, 10-30 parts of collagen synthesis-promoting peptides, and 5-8 parts of penetration-promoting peptides.

2. The composite polypeptide external dressing as described in claim 1, characterized in that: It includes the following raw materials in parts by weight: 30 parts of growth-promoting peptide, 25 parts of antibacterial and anti-inflammatory peptide, 20 parts of collagen synthesis-promoting peptide, and 7 parts of permeation-promoting peptide.

3. The composite polypeptide external dressing as described in claim 1, characterized in that: The proliferation-promoting peptide is a regeneration peptide from the stem tips of Dendrobium officinale, and its extraction method specifically includes: (1) Raw material pretreatment: Fresh Dendrobium officinale stem tip meristems were placed in a dark environment at 4℃ for 24-72 hours to induce culture and obtain pretreated raw materials; (2) Combined enzymatic hydrolysis: After the pretreated raw material is chopped, it is dispersed in deionized water at a material-to-liquid ratio of 1:(10-15 g / mL). A mixture of cellulase and pectinase is added at a mass ratio of 1:1-2, and the total amount added is 0.5%-1.0% of the dry weight of the raw material. The pH of the system is adjusted to 4.5-5.5, and the enzymatic hydrolysis is carried out at a constant temperature of 40-50℃ for 6-8 hours. (3) Enzyme inactivation and crude separation: After the enzymatic hydrolysis is completed, the temperature is raised to 95℃ and held for 10 minutes to inactivate the enzyme. After cooling, the supernatant is collected by centrifugation. Unhydrolyzed macromolecular polysaccharides and proteins are removed by ultrafiltration. The filtrate with a molecular weight of 800-1200 Da is collected, purified and dried to obtain white powdered Dendrobium officinale stem tip regeneration peptide with a purity of ≥90% and a molecular weight distribution of 800-1200 Da.

4. The composite polypeptide external dressing as described in claim 1, characterized in that: The antibacterial and anti-inflammatory peptides are derived from short disulfide-containing peptides obtained by enzymatic hydrolysis of byssal mucin from marine mollusks, with a molecular weight ≤1.2kDa. The extraction method specifically includes: (1) Raw material pretreatment: Take fresh mussel byssus, rinse to remove surface mud and microorganisms, cut into pieces and disperse in pH 7.0 Tris-HCl buffer at a material-to-liquid ratio of 1:(8-10) g / mL, add protease inhibitor, sonicate at 4℃, centrifuge, collect the precipitate, which is crude byssus mucin. (2) Targeted enzymatic hydrolysis: The crude byssal mucin was redispersed in deionized water at a substrate concentration of 3%-5% w / v. Papain was added at a concentration of 0.4%-0.6% of the substrate mass. The pH of the system was adjusted to 6.0-6.

5. The enzyme was hydrolyzed at 42-45℃ for 3-4 hours. After the enzymatic hydrolysis was completed, the temperature was raised to 80-100℃ and held for 8-12 minutes to inactivate the enzyme. After cooling, the supernatant was collected by centrifugation, ultrafiltration, purification, and freeze-drying to obtain a white powder.

5. The composite polypeptide external dressing as described in claim 1, characterized in that: The collagen synthesis promoting peptide is derived from bovine bone type I collagen. The extraction method is as follows: Bovine bone type I collagen is crushed, 5-8 times the amount of deionized water is added, the pH is adjusted to 7.0-7.5, 0.6-1.0% of collagen protein mass of protease is added for enzymatic hydrolysis for 2-4 hours, after enzyme inactivation, it is purified by 1000-2000 Da ultrafiltration membrane and dried to obtain glycine-proline enriched tripeptide.

6. The composite polypeptide external dressing as described in claim 1, characterized in that: The permeation-enhancing peptide is a menthol-peptide conjugate extracted from peppermint leaves. Its preparation method specifically includes: pulverizing peppermint leaves and passing them through a 60-80 mesh sieve; adding 10-15 times the weight of deionized water; adding 0.3%-0.8% of a compound enzyme (based on the weight of peppermint leaves); enzymatically hydrolyzing at 40-45℃ for 1.5-2.5 hours; the compound enzyme being a 1:1-3 mass ratio of cellulase and protease; centrifuging and collecting the supernatant to obtain a peppermint peptide solution; mixing the peppermint peptide solution with menthol at a mass ratio of 5-8:1; adding 0.1%-0.3% wt of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to the mixture; reacting at 30-35℃ for 1-3 hours; and then freeze-drying after adsorption through macroporous resin and filtration through a 300-800 Da ultrafiltration membrane to obtain the menthol-peptide conjugate.

7. The composite polypeptide external dressing as described in claim 1, characterized in that: It also includes 0.2-0.6 parts of natural antioxidants and 0.1-0.3 parts of metal ion chelating agents; the natural antioxidants are selected from tea polyphenols, rosmarinic acid or total asiaticosides, and the metal ion chelating agents are EDTA-2Na or sodium citrate.

8. The method for preparing a composite polypeptide topical dressing as described in claim 1, characterized in that: Includes the following steps: S1. Raw material pretreatment and dissolution: Weigh the proliferation-promoting peptide, antibacterial and anti-inflammatory peptide, collagen synthesis-promoting peptide, and permeation-promoting peptide, add sterile deionized water, and stir at low speed for 10-15 minutes at 25-30℃ until completely dissolved to obtain a single-component solution of each peptide. S2. Preparation of carrier matrix: Mix carboxymethyl chitosan and chondroitin sulfate at a mass ratio of (2-4):1, dissolve in deionized water at 40-50℃, and stir at 200-300 rpm until completely dissolved to form carrier mother liquor; S3. Assembly of the composite system: The single-component peptide solution obtained in S1 and the antioxidant and chelating agent are added sequentially to the carrier mother liquor in S2, ultrasonically dispersed for 15-25 minutes, pH adjusted to 6.8-7.2, and allowed to stand for cross-linking for 3-5 hours; S4. Sterilization and Packaging: The cross-linked system is sterilized by γ-ray irradiation and then packaged to obtain the composite polypeptide external dressing.

9. The method for preparing a composite polypeptide external dressing as described in claim 8, characterized in that: In step S1, the ratio of peptide to water in the liquid to liquid ratio is 1:10-15 (g:mL), and the stirring speed is 100-150 rpm. In step S3, the ultrasonic power is 400-600W, the static crosslinking temperature is 25-30℃, the solid content of the crosslinked system is 15-25wt%, and the pH value is 5.5-6.

5. The gamma ray irradiation dose in step S4 is 15-25 kGy, and the irradiation time is 10-20 minutes.

10. The use of a compound polypeptide topical dressing according to any one of claims 1-7 in the preparation of a drug for promoting skin wound healing.

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